Apr 27, 2020
Version 1
Isocitrate dehydrogenase V.1
- Kimberly Chapman1,
- Parith Wongkittichote2
- 1Children's National;
- 2Children's National, Department of Pediatrics, St. Louis Children’s Hospital, Washington University School of Medicine, St. Louis, MO, USA, Department of Pediatrics, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, 10400, Thailand
Protocol Citation: Kimberly Chapman, Parith Wongkittichote 2020. Isocitrate dehydrogenase. protocols.io https://dx.doi.org/10.17504/protocols.io.bfisjkee
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: April 24, 2020
Last Modified: April 27, 2020
Protocol Integer ID: 36146
Abstract
Isocitrate dehydrogenase is an enzyme which converts isocitrate to 2-oxoglutarate in the TCA cycle. IDH2 uses NADP and IDH3 uses NAD.
Our IDH2/3 assay examines accumulation of NADH/NADPH over time.
Reaction for IDH3, IDH2 uses NADP as its substrate and detected at OD 340 nm as well
Materials
- 0.5 M Potassium Phosphate Buffer pH 7.4 (room temp)
- 1 M MgCl2 (room temp)
- 100 mM NADP (-20) (made from β-Nicotinamide Adenine Dinucleotide Phosphate (CAS 53-59-8, kept at -20); dissolved in dW)
- 1 M Isocitrate (-20) (made from DL-isocitric acid (CAS 1637-73-6, kept at room temp); dissolved in dW)
Before start
Make sure have adequate amount of IDH buffer and that exactly pH 7.4 (best if prepared fresh with each assay)
Make sure that adequte mitochondrial lysate
Make sure spectrophotometer is set, wells labeled and plate ready to be read immediately
Prepare the IDH buffer new (Place on ice)
To make 10 mL IDH buffer
10 mM potassium phosphtate pH 7.4 (200 μL 0.5 M potasium phosphate pH 7.4),
2 mM MgCl2 (20μL 1M MgCl2),
1mM NADP (100 μL 100mM NADP)
5mM isocitrate (50 μL 1M isocitrate)
Add water to make 10 mL after pH ing to 7.4
Prepare mitochondrial lysates per protocol (will need 15-30 μg in 20 μL)
Load 20 μL into 96 well plate in triplicate
Set spectrophotometer to Kinetic, dual wavelength 340, 380 nm, 30 reads, interval 30 sec at 30°C
Add 200 μL of IDH buffer to each well of plate (use a multi-chamber pipetter) sitting on the holder for the spectrometer and mix by pipetting in and out 2x and start spectrophotometer
Calculate relative activity from the values of OD340